bs_bs_banner

Research Paper

Journal of Pharmacy
And Pharmacology

Cross-linked human serum albumin dimer has the potential
for use as a plasma-retaining agent for the fatty
acid-conjugated antidiabetic drugsaFaculty of Pharmaceutical Sciences and gDDS Research Institute, Sojo University, cDepartment of Biopharmaceutics and eCenter for ClinicalPharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, dSchool of Health Sciences, Faculty of
Medicine, Kanazawa University, Kanazawa, fDepartment of Applied Chemistry, Faculty of Science and Engineering, Chuo University, Bunkyo-ku,
Japan and bSchool of Pharmacy, Faculty of Health Sciences, Curtin University, Perth, Western Australia, Australia

Keywords
albumin; diabetes; fatty acid; glucagon like
peptide-1 receptor agonist; insulin analogue
Correspondence
Masaki Otagiri, Faculty of Pharmaceutical
Sciences, Sojo University, 4-22-1 Ikeda,
Nishi-ku, Kumamoto 862-0082, Japan.
E-mail: otagirim@ph.sojo-u.ac.jp
Received July 14, 2014
Accepted September 28, 2014
doi: 10.1111/jphp.12338

Abstract
Objectives The half-life of fatty acid-conjugated antidiabetic drugs are prolonged
through binding to albumin, but this may not occur in diabetic patients withnephropathy complicated with hypoalbuminemia. We previously showed thathuman serum albumin (HSA) dimerized at the protein’s Cys34 by 1,6bis(maleimido)hexane has longer half-life than the monomer under high permeability conditions. The aim of this study was to investigate the superior ability ofthis HSA dimer as a plasma-retaining agent for fatty acid conjugated antidiabetic
drugs.
Methods The diabetic nephropathy rat model was prepared by administering asingle injection of streptozotocin (STZ) intravenously, and the pharmacokinetic
properties of HSA monomer and dimer were evaluated. Site-specific fluorescentprobe displacement experiments were performed using warfarin anddansylsarcosine as site I and site II specific fluorescent probes, respectively.
Key findings The half-life of the HSA dimer in STZ-induced diabetic nephropathy model rats was 1.5 times longer than the HSA monomer. The fluorescentprobe displacement experiment results for HSA monomer and dimer were similar,
where fatty acid-conjugated antidiabetic drugs displaced dansylsarcosine but not
warfarin in a concentration-dependent manner.
Conclusions The HSA dimer shows potential for use as a plasma-retaining agent
for antidiabetic drugs due to its favourable pharmacokinetic properties.

Introduction
There were approximately 285 million cases of diabetes
which is equivalent to ∼6.4% of the world population
making it one of the most common diseases in the world,[1]and as the disease progresses, diabetic patients are commonly associated with complications such as diabetic
polyneuropathy, retinopathy, nephropathy and angiopathy.
New classes of medicines such as dipeptidyl peptidase-4
inhibitors, glucose transporter inhibitors and insulin preparations have been developed to better control the blood
sugar level as well as to improve quality of life. Among thesenew drugs, insulin analogues (insulin detemir, insulin
degludec) and glucagon-like peptide-1 (GLP-1) receptor

agonist (liraglutide), have gained a place in diabetic treatment worldwide recently. These drugs reversibly bindalbumin through the long-chain fatty acid attached to them(insulin or mutated GLP-1 with 97% homology to native
GLP-1). As a result, they distribute more slowly fromthe injection site to peripheral target tissues thus extending their half-life[2–4] (insulin detemir; 5–7 h, insulin
degludec; <24 h vs native insulin; 4–6 min, liraglutide;
13 h vs GLP-1; <2 min) and preventing them from degradation in circulation as well as avoiding glomerulus filtration.[3] These properties allow them for once or twice-daily
dosing.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

255

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

Kazuaki Taguchia, Victor Tuan Giam Chuangb, Keishi Yamasakia,g, Yukino Uratac, Ryota Tanakac,
Makoto Anrakua, Hakaru Seoa,g, Keiichi Kawaid, Toru Maruyamac,e, Teruyuki Komatsuf and
Masaki Otagiria,c,g

Potential of albumin dimer in diabetes

Kazuaki Taguchi et al.

Materials and Methods

from Wako Pure Chemical Industries Ltd (Tokyo, Japan)
and used without further purification. BMH was purchasedfrom Thermo Fisher Scientific, Inc. (Rockford, IL, USA).
111
InCl3 (74 MBq/ml in 0.02 N HCl) was donated by Nihon
Medi-Physics (Takarazuka, Japan). STZ was purchased fromNakarai Tesque (Lot. M3M2485, Kyoto, Japan). All otherchemicals were of the highest grade commercially available,
and all solutions were prepared using deionized distilledwater. The HSA dimer was synthesized according to procedures we have reported previously.[8]Preparation of streptozotocin-induced
diabetic nephropathy model rat
All animal experiments were performed according to theguidelines, principles and procedures for the care and use oflaboratory animals of Kumamoto University (Approval
No.C20-206). Sixteen male Sprague–Dawley (SD) rats (5weeks old, Kyudou Co., Kumamoto, Japan) were used inthis study. All animals were maintained under conventional
housing conditions, with food and water ad libitum, in atemperature-controlled room with a 12-h dark/light cycle.
A single dose of STZ (60 mg/kg) given intravenously torats has been reported to produce clear diabetic symptoms,[11] hence 11 SD rats were administered STZ at a doseof 60 mg/kg through the tail vain under ether anaesthesia.
Five SD rats were used as control group.

Assessment of diabetic nephropathy model
rat induced by streptozotocin
One day before the experiments (13 days after STZ administration), urine was collected for 24 h in a metabolic cage,
and the volume of urine was measured. After urine collection was completed (14 days after STZ administration), five
STZ-induced diabetic nephropathy model rat and fivecontrol rats were anaesthetized with ether, and blood
samples were collected from the inferior vena cava. The
blood samples were centrifuged (3000 g, 10 min) to obtainplasma for the analysis of serum creatinine (Scr) and bloodurea nitrogen (BUN) and glucose. Mean Scr, BUN andglucose concentrations were measured by LaboAssay
Creatinine, UNB-test Wako and GlucoseCII-test Wako
(Wako Pure Chemical Industries, Ltd, Osaka, Japan) assays,
respectively. After blood samples were collected, the rats
were sacrificed by acute bleeding from the abdominal aorta
and to obtain the kidney and pancreas for histopathologicalstudies. The organs were fixed in 4% paraformaldehyde
overnight.

ChemicalsHistopathological examination

HSA (Lot. U389PX, 25% wt.) was obtained from BENESIS(Osaka, Japan). Ethanol and dithiothreitol were purchased

The fixed kidneys and pancreases were embedded inparaffin, sectioned into 5-μm slices and then stained with256

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

Clinically, more than 40% of diabetic patients have diabetic nephropathy. As the diabetic nephropathy progressed
to end-stage renal failure, continuous protein leakage
through glomerulus and high capillary protein permeability
took place, accompanied by an increased albumin flux
to the extravascular compartment. Consequently, patientswith diabetic nephropathy will also be affected byhypoalbuminemia at the same time. Hence, the half-lives offatty acid-conjugated insulin analogues and GLP-1 receptor
agonist may be shorter in diabetic nephropathy patientscomplicated with hypoalbuminemia, which may result ininadequate blood sugar control. Although human serum
albumin (HSA) infusion may be a therapeutic option forthese patients, the infused albumin is expected to rapidly
distribute to the extravascular compartment due to highcapillary protein permeability and increased glomerulus filtration. Therefore, a new albumin preparation that canmaintain albumin concentration in the circulation ofpatients with diabetic nephropathy and also preserves drug
binding capacity is highly desirable.
In general, increasing the molecular size of albumin bypolymerization and PEGylation and others is widely recognized as the best strategy for reducing glomerulus filtrationand high capillary permeability.[5–7] Although these measures could prolong the blood retention of albumin polymers, the drug binding capacity HSA is compromised. We
previously reported HSA dimer prepared by cross-linking
two HSA molecules with 1,6-bis(maleimido)hexane(BMH)[8] showed superior plasma retention property compared with HSA monomer in the doxorubicin-induced
nephrotic rat model.[9] In addition, the length of BMH
seems to be sufficiently long (16.1 Å), permitting HSA
dimer to preserve the structural properties of native HSA
monomer including ligand binding capacity and bloodcompatibility.[8,10] These evidence had led us to hypothesizethat HSA dimer produced from two HSA molecules chemically linked with BMH has the potential as a plasmaretaining agent of antidiabetic drugs, such as fatty acidconjugated insulin analogues and GLP-1 receptor agonist,
for treating hypoalbunemia under diabetic nephropathy
condition. A rat model of diabetic nephropathy induced bytreatment with streptozotocin (STZ) was used to evaluate
the pharmacokinetic properties of HSA monomer and HSA
dimer. In addition, the binding ability of HSA dimer for
fatty acid-conjugated insulin analogues (insulin detemir
and insulin degludec) and GLP-1 receptor agonist(liraglutide) was determined.

Kazuaki Taguchi et al.

Periodic acid-Schiff (PAS) and H&E, respectively on a slide.
The slide was observed using Microscope (Keyence,
BZ-8000, Osaka, Japan).

Proteins labeling with 111InThe pharmacokinetic experimental
protocol in streptozotocin-induced diabetes
model rat
Fourteen days after STZ administration, the rats were used
as a STZ-induced diabetic nephropathy model rat. All STZinduced diabetic nephropathy model rat were anaesthetizedusing ether and received a single injection of 111In-labeled
HSA monomer in saline (n = 3, 1 mg/kg, 2 ml/kg,
1 × 107 cpm/kg) or HSA dimer in saline (n = 3, 1 mg/kg,
2 ml/kg, 1 × 107 cpm/kg) via the tail vein. At each time
point (3 min, 10 min, 30 min and 1, 2, 6, 14, 18, 24, 30, 38,
42 and 48 h) after an injection of the 111In-labeled protein, a
100 μl aliquot of blood was collected from the tail vein, andplasma was obtained by centrifugation (6000 g, 5 min). At
48 h after the injection of the 111In-labeled protein, the rats
were sacrificed, and the organs were collected and rinsed
with saline. Urine was collected at fixed intervals in a metabolic cage. The levels of 111In in the plasma, excised organs
and urine were determined using a gamma counter (ARC5000; Aloka, Tokyo, Japan).

Fluorescent probe
displacement experiments
Warfarin potassium (WF) (Eisai Co., Tokyo, Japan) anddansylsarcosine (DNSS) (Sigma, St. Louis, MO, USA) wereused as the fluorescent probes for site I and site II, respectively.[12] Both probes were dissolved in 100% ethanol. The
displacement experiments of WF and DNSS by insulin analogues (insulin detemir; Levemir (novo nordisk, Bagsvaerd,
Denmark), insulin degludec; Tresiba (novo nordisk,
Bagsvaerd, Denmark)) and GLP-1 receptor agonist(liraglutide; Victoza (novo nordisk, Bagsvaerd, Denmark))
were performed by recording the fluorescence of a solution
containing 2 μm WF (or DNSS) and 2 μm HSA monomer(or 1 μm HSA dimer) while gradually increasing the insulin
analogues (or GLP-1 receptor agonist) concentration. The
excitation wavelengths for WF and DNSS were 320 and
350 nm, respectively. The emission spectra for WF andDNSS were recorded in the range of 350–450 nm and 400–
600 nm, respectively.

Data analysis
Pharmacokinetic analyses after the administration of 111Inlabeled HSA monomer or dimer were carried out using a
two-compartment model. Pharmacokinetic parameters were
calculated by curve fitting using a nonliner least squares
program (MULTI) for microcomputers (http://www.pharm.kyoto-u.ac.jp/byoyaku/Kinetics/download.html).[13] Data areshown as the mean ± SD for the indicated number ofanimals. Significant differences among each group were
determined using the two-tail unpaired Student’s t-test. A
probability value of P < 0.05 was considered to be statisticallysignificant.

Results
Assessment of a rat model of diabetes
induced by streptozotocin
To evaluate the extent of pancreas impairment of the STZtreated rats, we measured the changes of body weight, the24-h urine collection volume, the levels of glucose inplasma. As a result, the STZ-treated rats showed bodyweight loss (307.5 ± 9.6 and 251.3 ± 20.9 g, P < 0.01, forcontrol and STZ-treated rat, respectively, Figure 1a) andincreased volume of 24-h urine collection (34.5 ± 11.6 and109.5 ± 36.0 ml/day, P < 0.01, for control and STZ-treatedrat, respectively, Figure 1b) at 14 days after STZ treatment
when compared with the control rats. The glucose levels in
the STZ-treated rats were significantly increased by approximately 1.5-fold compared with that in the control rats
(194.2. ± 2.3 and 301.6 ± 90.1 mg/dl, P < 0.05, for control
and STZ-treated rat, respectively, Figure 1c). Furthermore,degenerative changes and clear cytoplasm in the pancreatic
islet of the STZ-treated rats was observed from the H&E
staining data, but not in the control rats (Figure 1d).
Scr and BUN levels were measured to determine thedegree of renal impairment of the STZ-treated rats. The Scr
and BUN levels were not significantly different between
control and STZ-treated rats (Figure 1e and 1f). However,glomerulus damage was clearly observed in the STZ-treatedrats from the PAS staining data, but not in the control rats
(Figure 1g).

Pharmacokinetic experiment in
streptozotocin-induced diabetes model rat
Pharmacokinetic properties of the HSA monomer and dimer
were examined in the STZ-induced diabetic nephropathy
model rats. The volume of 24-h urine collection in STZinduced diabetic model rats used in the pharmacokinetic
study was 122.0 ± 22.8 ml (monomer; 113.8 ± 24.6 ml, dimer;
130.2 ± 22.2 ml, not statistically significant difference).
Figure 2a shows the plasma concentration time course of
the 111In-HSA monomer and 111In-HSA dimer administered© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

257

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

For the pharmacokinetic experiments, the HSA monomer
and dimer were radiolabeled with 111In as previously
reported.[9] Both 111In-labeled HSA monomer and dimer
were mixed with unlabeled HSA monomer and dimer,
respectively, to adjust the protein concentration before use
in the pharmacokinetic experiments.

Potential of albumin dimer in diabetes

Potential of albumin dimer in diabetes

Kazuaki Taguchi et al.

200
150
100
50
0

Control

STZ

Control

(d)

(f)
1.2

25

1.0

20
BUN (mg/dL)

Scr (mg/dL)

Control

STZ

Control

STZ

450
400
350
300
250
200
150
100
50
0
Control

STZ

STZ

(e)

0.8
0.6
0.4

15
10
5

0.2
0

**
glucose (mg/dL)

**

250

160
140
120
100
80
60
40
20
0

0
Control
Control

STZ
STZ

Figure 1 The parameters (a) body weight, (b) urine volume and (c) glucose in plasma in control rats (opened column) and streptozotocin-treatedrats (closed column). (d) Light micrographs of pancreas stained with H&E. The parameters of (e) serum creatinine and (f) blood urea nitrogen inplasma in control rats (opened column) and streptozotocin-treated rats (closed column). (g) Light micrographs of kidney stained with Periodic acidSchiff stain. The Sprague–Dawley rats were administered streptozotocin as a single injection (60 mg/kg) through the tail vain. Urine was starting to
be collected for 24 h in a metabolic cage at 13 days after streptozotocin administration. Fourteen days after the streptozotocin injection, biochemical parameters and histological study were evaluated. The values are the mean ± SD. (n = 5) *P < 0.05 vs control rats, **P < 0.01 vs control rats.

to the STZ-induced diabetic nephropathy model rats at adose of 1 mg/kg, and Table 1 lists the pharmacokinetic
parameters obtained using a two-compartment model. The
plasma concentration of HSA monomer was rapidly cleared
compared with that of the HSA dimer in diabetic neph258

ropathy rats (Figure 2a). The clearance for the HSA dimer
decreased to half of the HSA monomer (0.61 ± 0.07 and0.34 ± 0.06 ml/h, P < 0.01, for the HSA monomer anddimer, respectively). The t1/2 and the area under theconcentration-time curve (AUC) of the HSA dimer© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

Body weight (g)300

Urine volume (mL/day)

350

(g)

(c)

(b)

(a)

Kazuaki Taguchi et al.

Potential of albumin dimer in diabetes

10

1
monomer
dimer
0.1

0

12

24

36

48 (hr)(b)
monomer
dimer
15

**
**

10
5

ey
liv
sp er
le
en
lu
ng
pa heanc rt
sm st rea
al om sla l in ac
rg te h
e sti
in n
te e
st
in
m e
us
cl
e
sk
in
br
ai
n
te
st
is

0

ki

dn

Organ level in whole tissue(% of injection of dose)20

increased compared with the HSA monomer (t1/2, 14.1 ± 1.2
and 21.1 ± 1.8 h, P < 0.01 and AUC, 165 ± 19 and298 ± 49 h % of dose/ml, P < 0.01, for the HSA monomer
and dimer, respectively) (Table 1).
The tissue distribution (kidney, liver, spleen, lung, heart,pancreas, stomach, small intestine, large intestine, muscle,skin, brain and testis) of the 111In-HSA monomer and 111InHSA dimer at 48 h after administration was also examined.
As shown in Figure 2b, both HSA monomer and dimer
mainly distributed to the liver and muscle. The accumulation of HSA dimer in muscle was lower compared with the
HSA monomer (14.8 ± 2.1 and 10.5 ± 0.1% of dose,P < 0.01, for the HSA monomer and dimer, respectively),but HSA dimer in the liver was significantly higher than
HSA monomer (10.0 ± 0.3 and 14.3 ± 0.03% of dose,P < 0.01, for the HSA monomer and dimer, respectively).
There were no significant differences between HSA
monomer and dimer in other organs (Figure 2b). In addition, the radioactivity of 111In after administration of the
HSA monomer and dimer were approximately 8% and 5%of dose at 48 h, respectively.

Figure 2 (a) Plasma concentration of 111In-HSA monomer (opencircles) and dimer (closed circles) after intravenous administration to
streptozotocin-induced diabetic nephropathy model rats. (b) Tissue distribution of radioactivity in each whole tissue (% of injection ofdose) at 48 h after intravenous administration of 111In-HSA monomer(open bars) or dimer (closed bars) to streptozotocin-induced diabetic
model rats. Fourteen days after the streptozotocin injection, allstreptozotocin-induced diabetic nephropathy model rats received asingle injection of 111In-HSA monomer or dimer at a dose of 1 mg/kg.
Each point represents the mean ± SD. (n = 3) **P < 0.01 vs humanserum albumin monomer.

Table 1 Pharmacokinetic parameters after administration of 111Inhuman serum albumin monomer and dimer to diabetic nephropathy
model rats
parameters111

t1/2 (h)AUC (h×% of dose/ml)ke (hr-1)
CL (ml/h)
Vd (ml)MRT (h)14.1 ± 1.2
165 ± 190.076 ± 0.005
0.61 ± 0.07
11.6 ± 1.1
18.4 ± 1.3

In-HSA monomer

111

In-HSA dimer

21.1 ± 1.8**298 ± 49**0.050 ± 0.006**
0.34 ± 0.06**
9.8 ± 1.8
29.2 ± 2.1**

AUC, area under the concentration-time curve; CL, clearance; HAS,
human serum albumin; MRT, mean residence time; t1/2, half-life; Vd,
volume of distribution at steady state. Each value represents themean ± SD. (n = 3) **P < 0.01 vs 111In-HSA monomer.

Fluorescent probe
displacement experiments
To function as a retaining agent for fatty acid conjugatedinsulin analogues and GLP-1 receptor agonist, HSA dimer
must maintain similar ligand binding capacity as HSA
monomer. Therefore, site-specific fluorescent probes displacement experiments in HSA monomer and dimer were
performed using WF and DNSS as site I and site II-specificfluorescent probes, respectively. Addition of fatty acidconjugated insulin analogues and GLP-1 receptor agonist to
the HSA proteins–fluorescent probes mixtures caused aconcentration-dependent reduction in the DNSS fluorescence intensity but did not cause a reduction of the WF
fluorescence intensity in both HSA monomer and dimer(Figure 3).

Discussion
The results in this study showed that HSA dimer has alonger blood retention property compared with HSA
monomer in the diabetic nephropathy rat model, possibly
through suppression of the infused HSA dimer flux to theextravascular compartment (Figure 2). In addition, HSA
dimer has identical ligand binding capacity for the fatty acidconjugated insulin analogues and GLP-1 receptor agonist as
the HSA monomer (Figure 3). These findings suggested that
HSA dimer produced by linking two HSA molecules with
BMH chemically can function as a plasma-retaining agentof the fatty acid conjugated antidiabetic drugs under diabetic nephropathy condition.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

259

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

Plasema level(% of injection of dose/mL)(a)

Potential of albumin dimer in diabetes

Kazuaki Taguchi et al.

(a) Insulin detemir
120

120
100
% of initial

80
60
40

80
60
40
Warfarin

Warfarin20020Dansylsarcosine
0

2

4

6

8

10

0

12 (µM)

Dansylsarcosine
0

2

4

6

8

10

12 (µM)810 (µM)

100

100

80

80

% of initial% of initial(b) Insulin degludec

60
40
Warfarin
20

60
40
Warfarin

20

Dansylsarcosine

Dansylsarcosine

0

0
0

2

4

6

8

10 (µM)

0

2

4

6

100

100

80

80

% of initial% of initial(c) Liraglutide

60
40
Warfarin

20

60
40
Warfarin

20

Dansylsarcosine

Dansylsarcosine

0

0
0

5

10 15 20 25 30 35 (µM)

05

10 15 20 25 30 35 (µM)

Figure 3 Displacement experiments in human serum albumin monomer (right panel) and human serum albumin dimer (left panel) for warfarin
potassium (open circles) and dansylsarcosine (closed circles) by (a) insulin detemir, (b) insulin degludec and (c) liraglutide. Displacement experiments
for warfarin potassium and dansylsarcosine by insulin analogues and glucagon-like peptide-1 (GLP-1) receptor agonist were performed by recording
the fluorescence of a solution containing 2 μM warfarin potassium (or 2 μM dansylsarcosine) and 2 μM human serum albumin monomer (or 1 μM
human serum albumin dimer). The excitation wavelengths for warfarin potassium and dansylsarcosine were 320 and 350 nm, respectively. The emission spectra for warfarin potassium and dansylsarcosine were recorded in the range of 350–450 nm and 400–600 nm, respectively.

STZ-induced diabetic mellitus animals were widely usedin experiments that evaluate efficacy of antidiabetic agents.
Thus, we evaluated the blood retention property of HSA
dimer in STZ-induced diabetic nephropathy model rats.
The t1/2 for the HSA dimer was 1.5 times longer than that of
the HSA monomer in STZ-induced diabetic nephropathyrat (Figure 2, Table 1). In addition, the mean resident timefor HSA dimer was significantly longer than that of the
HSA monomer (Table 1), which implied that HSA dimer
had a prolonged retention in blood circulation as compared260

with HSA monomer. It is well accepted that glucose toxicity
induces barrier dysfunction via direct targeting of theexchange microvascular endothelium that ultimately lead toincreased extravasation of plasma fluid and proteins,
including albumin.[14] In fact, leakage of exogenously
infused macromolecules, such as albumin and dextran,
from vascular space to extravascular space were detected in
the diabetic model rats.[15,16] In a previous study, Matsushita
et al. reported that the t1/2 of genetically fused HSA dimer
was prolonged by 1.1 times due to a decrease in the vascular© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

% of initial100Kazuaki Taguchi et al.

with relatively moderate uptake by the liver was seen forproteins of intermediate size.[20] Therefore, higher hepatic
distribution of HSA dimer than HSA monomer could be
due to the increased molecular size of HSA via chemical
dimerization. This is also supported by our previous
pharmacokinetic study using normal rats that showed sametendency as in this study.[9]To function as a retaining agent of fatty acid-conjugatedinsulin analogues and GLP-1 receptor agonist, HSA dimer
must preserve the binding capacity of native HSA. These
drugs can reversibly bind albumin through the long-chain
fatty acid (myristyl, hexadecandioyl and palmitoyl fatty acidfor insulin detemir, insulin degludec and liraglutide, respectively) that attached via a spacer to an amino acid in insulin
or mutated GLP-1.[2,3] In this study, the fluorescent probe
displacement experiment results for HSA monomer anddimer were similar, where insulin analogues and GLP-1
receptor agonist displaced DNSS but not WF in aconcentration-dependent manner (Figure 3).
X-ray crystallographic studies revealed that HSA has
seven fatty acid binding sites.[21] In addition, the fatty acidbinding affinity (high/low) of each site has been also identified with 13C nuclear magnetic resonance spectroscopy[22,23]
and site-directed mutagenesis of HSA.[24] These studies
identified that fatty acid binding sites 3 and 4, which correspond to a major drug binding site II, are high-affinity fatty
acid binding sites. On the other hand, site I is also found to
be a location for another one of the fatty acid binding sites.
Based on molecular dynamics simulations reported by
Fujiwara et al., fatty acids preferentially bound to highaffinity fatty acid binding sites 2, 4 and 5 in the presence ofWF.[25,26] This may explain insulin analogues and GLP-1
receptor agonist would only displace DNSS. These results
suggest that the binding site and binding capacity of HSA
monomer were preserved in the HSA dimer. In fact, a previous study showed that the binding constants of WF anddiazepam for the chemically linked HSA dimer were similar
to those for the HSA monomer.[8]Although this study demonstrated the utility of HSA
dimer as a plasma–retaining agent for the fatty acidconjugated antidiabetic drugs, some limitations must also
be taken into account. The dose used in this study (1 mg/kg) was less than a pharmacological dose in clinical situation. Therefore, a pharmacokinetic examination of HSA
monomer and dimer at pharmacological doses should be a
subject of future investigation. Another major point ofexperimental limitation noted in this study is that there wasno direct evidence that HSA dimer can work as a plasmaretaining agent for the fatty acid-conjugated antidiabetic
drugs in vivo. In the presence of endogenous albumin invivo, these fatty acid-conjugated antidiabetic drugs wouldinteract with not only HSA dimer but also endogenous
albumin (monomer), and possibly other fatty acid binding© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

261

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

permeability of the HSA dimer compared with the HSA
monomer in carrageenin-air-pouch rats.[7] Therefore,
increasing the molecular size of HSA via chemical
dimerization is thought to have retarded HSA dimer
extravasation through the vascular endothelium, resulting
in longer plasma retention in this study. In fact, in the diabetic model rats, the distribution of HSA dimer in muscle(10.5 ± 0.1% of dose) was also less than HSA monomer(14.8 ± 2.1% of dose) (Figure 2b).
The glomerular biological membrane has properties that
do not allow larger proteins such as albumin to be filtered.
This restriction can be explained by several mechanisms,
including charge repulsion in the glomerular basement
membrane and barrier.[17] As diabetes progresses, functionalor structural glomerular alterations eventually will occur.
Satoh et al. reported the increased glomerular permeabilityof 40 kDa rhodamine-labeled dextran in STZ-induced diabetic rats compared with normal rats.[18] Therefore, it isanticipated that increasing the molecular size of HSA by
dimerization might prevent filtration by the renal glomerulus, results in a longer lifetime in blood circulation.
However, there was no significant difference in distribution
in kidney between HSA monomer and dimer in this study(Figure 2b). Diabetic nephropathy is classified into fivestages (stages I–V) depending on the progression of renal
failure, massive albuminuria can be observed in advanced
diabetes (stages III–V).[19] To evaluate the progression ofdiabetic nephropathy, we determine the urinary protein
concentration in the rat model of diabetic nephropathy at
14 days after STZ administration. The urinary protein in
the diabetes model rat was 12.7 ± 3.5 mg/day, which wasmuch less than that of the nephritic rats induced bydoxorubicin (213 ± 28 mg/day) but was close to that ofnormal rats (8.0 ± 3.4 mg/day).[9] In addition, the urinary
excretion of the HSA monomer and dimer in STZ-induced
diabetic nephropathy rat were similar to those in normal
rat.[9] Furthermore, although glomerulus damage wasobserved in the STZ-induced diabetic nephropathy model
rats from the PAS staining (Figure 1g), Scr and BUN concentrations in the STZ-induced diabetic nephropathy
model rats were at about the same level as those of thecontrol rats (Figure 1e and 1f). These results suggested thatthe extent of diabetic nephropathy did not progress to endstage renal failure in the diabetic nephropathy rat used inthis study. Thus, this may explain no difference in the distribution in kidney between HSA monomer and dimer.
Although 111In-HSA dimer showed a similar tendency oftissue distribution with that of the 111In-HSA monomer, theextent of accumulation of the HSA dimer in the liver wasmuch greater than that of 111In-HSA monomer (Figure 2b).
Rennen et al. previously reported that a rapid blood clearance with a predominant uptake by the liver was found forthe largest proteins, while a relatively slow blood clearance

Potential of albumin dimer in diabetes

Potential of albumin dimer in diabetes

Kazuaki Taguchi et al.

proteins, too. Therefore, it is too difficult to obtain direct
evidence showing HSA dimer can work as a plasmaretaining agent in vivo. However, this could be a subject offurther study in future.

or in advanced clinical trials,[29,30] HSA dimer is expected tofurther expand the application possibility for use in various
clinical conditions.

Declarations
Conflict of interest

The BMH-linked HSA dimer shows potential for use as aplasma-retaining agent for the fatty acid-conjugatedantidiabetic drugs due to its favourable pharmacokinetic
properties. Furthermore, the chemical and genetic HSA
dimers have been reported to have potential for use as adrug carrier, novel plasma expander and artificial oxygen
carrier because of their physicochemical properties andpharmacokinetic properties.[7,9,27,28] Since there are a numberof HSA-conjugated drugs that have already been marketed

The Author(s) declare(s) that they have no conflicts ofinterest to disclose.

References
1. Elsadek B, Kratz F. Impact of albuminon drug delivery – new applications
on the horizon. J Control Release 2012;
157: 4–28.
2. Gough SC et al. Insulin degludec:
overview of a novel ultra long-acting
basal insulin. Diabetes Obes Metab
2013; 15: 301–309.
3. Ahren B, Burke B. Using albumin to
improve the therapeutic properties ofdiabetes treatments. Diabetes Obes
Metab 2012; 14: 121–129.
4. Deacon CF et al. Both subcutaneously
andintravenously
administeredglucagon-like peptide I are rapidly
degraded from the NH2-terminus in
type II diabetic patients and in healthy
subjects. Diabetes 1995; 44: 1126–
1131.
5. Zhao T et al. Site-specific chemical
modification of human serum
albumin with polyethylene glycol prolongs half-life and improves intravascular retention in mice. Biol Pharm
Bull 2012; 35: 280–288.
6. Komatsu T et al. Albumin clusters:
structurally defined protein tetramer
and oxygen carrier including thirtytwo iron(II) porphyrins. Biomacromolecules 2005; 6: 3397–3403.
7. Matsushita S et al. Recombinanthuman serum albumin dimer has highblood circulation activity and low vascular permeability in comparison with

262

Funding
This research was partially supported by Grant-in-Aid for
Scientific Research (no. 20350058) from JSPS, and Grantin-Aid for Scientific Research for Priority Area (area 2107)
from MEXT Japan, and Health Science Research Grants(Regulatory Science) from MHLW Japan.

native human serum albumin. PharmRes 2006; 23: 882–891.
8. Komatsu T et al. Physicochemical
characterizationofcross-linked
human serum albumin dimer and its
synthetic heme hybrid as an oxygen
carrier. Biochim Biophys Acta 2004;
1675: 21–31.
9. Taguchi K et al. Superior plasma
retention of a cross-linked humanserum albumin dimer in nephrotic
rats as a new type of plasma expander.
Drug Metab Dispos 2010; 38: 2124–
2129.
10. Taguchi K et al. Pharmaceuticalaspects of the recombinant humanserum albumin dimer: structuralcharacteristics, biological properties,
and medical applications. J Pharm Sci
2012; 101: 3033–3046.
11. Elias D et al. Autoimmune diabetes
induced by the beta-cell toxin STZ.
Immunity to the 60-kDa heat shockprotein and to insulin. Diabetes 1994;
43: 992–998.
12. Panjehshahin MR et al. Effect ofvalproic acid, its unsaturated metabolites and some structurally related
fatty acids on the binding of warfarinand dansylsarcosine to humanalbumin. Biochem Pharmacol 1991; 41:
1227–1233.
13. Yamaoka K et al. A pharmacokinetic
analysis program (multi) for microcomputer. J Pharmacobiodyn 1981; 4:
879–885.

14. Yuan SY et al. Microvascular permeability in diabetes and insulin resistance. Microcirculation 2007; 14: 363–
373.
15. Beals CC et al. Microvascular clearance of macromolecules in skeletal
muscle of spontaneously diabetic rats.
Microvasc Res 1993; 45: 11–19.
16. Yamaji T et al. Increased capillary permeability to albumin in diabetic rat
myocardium. Circ Res 1993; 72: 947–
957.
17. Haraldsson B et al. Properties of theglomerular barrier and mechanisms ofproteinuria. Physiol Rev 2008; 88: 451–
487.
18. Satoh M et al. In vivo visualization ofglomerular microcirculation andhyperfiltration in streptozotocininduced diabetic rats. Microcirculation
2010; 17: 103–112.
19. Macisaac RJ, Jerums G. Diabetic
kidney disease with and without albuminuria. Curr Opin Nephrol Hypertens
2011; 20: 246–257.
20. Rennen HJ et al. The effect of molecular weight on nonspecific accumulation of (99m)T-labeled proteins ininflammatory foci. Nucl Med Biol
2001; 28: 401–408.
21. Bhattacharya AA et al. Crystallographic analysis reveals common
modes of binding of medium andlong-chain fatty acids to humanserum albumin. J Mol Biol 2000; 303:
721–732.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

Conclusion

Kazuaki Taguchi et al.

allosteric effects of fatty acids on thebinding of warfarin to human serum
albumin revealed by molecular
dynamics and free energy calculations.
Chem Pharm Bull (Tokyo) 2011; 59:
860–867.
26. Fujiwara S, Amisaki T. Fatty acidbinding to serum albumin: molecular simulation approaches. Biochim
Biophys Acta 2013; 1830: 5427–
5434.
27. Ishima Y et al. S-Nitrosated humanserum albumin dimer is not only anovel anti-tumor drug but also a
potentiator for anti-tumor drugs withaugmented EPR effects. Bioconjug
Chem 2012; 23: 264–271.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 255–263

28. Komatsu T et al. Cross-linked humanserum albumin dimer incorporating sixteen (tetraphenylporphinato)iron(II) derivatives: synthesis, characterization, and O2-binding property.
Macromolecules 1999; 32: 8388–8391.
29. Kratz F. A clinical update of using
albumin as a drug vehicle – a commentary. J Control Release 2014; 190:
331–336.
30. Kratz F. Elsadek B. Clinical impact ofserum proteins on drug delivery.
J Control Release 2012; 161: 429–
445.

263

Downloaded from https://academic.oup.com/jpp/article/67/2/255/6128115 by Kanazawa University Library user on 13 January 2022

22. Simard JR et al. Locating high-affinity
fatty acid-binding sites on albumin by
x-ray crystallography and NMR spectroscopy. Proc Natl Acad Sci U S A
2005; 102: 17958–17963.
23. Simard JR et al. Location of highand low affinity fatty acid bindingsites on human serum albumin
revealed by NMR drug-competition
analysis. J Mol Biol 2006; 361: 336–
351.
24. Kragh-Hansen U et al. Chain lengthdependent binding of fatty acidanions to human serum albuminstudied by site-directed mutagenesis.
J Mol Biol 2006; 363: 702–712.
25. Fujiwara S, Amisaki T. Steric andPotential of albumin dimer in diabetes